U.S. patent number 5,439,881 [Application Number 08/049,783] was granted by the patent office on 1995-08-08 for gene encoding nematode-active toxin ps63b cloned from bacillus thuringiensis isolate.
This patent grant is currently assigned to Mycogen Corporation. Invention is credited to Kenneth E. Narva, Jewel M. Payne, George E. Schwab.
United States Patent |
5,439,881 |
Narva , et al. |
* August 8, 1995 |
**Please see images for:
( Certificate of Correction ) ** |
Gene encoding nematode-active toxin PS63B cloned from Bacillus
thuringiensis isolate
Abstract
This invention concerns genes or gene fragments which have been
cloned from novel Bacillus thuringiensis isolates which have
nematicidal activity. These genes or gene fragments can be used to
transform suitable hosts for controlling nematodes.
Inventors: |
Narva; Kenneth E. (San Diego,
CA), Schwab; George E. (La Jolla, CA), Payne; Jewel
M. (San Diego, CA) |
Assignee: |
Mycogen Corporation (San Diego,
CA)
|
[*] Notice: |
The portion of the term of this patent
subsequent to August 14, 2007 has been disclaimed. |
Family
ID: |
27374822 |
Appl.
No.: |
08/049,783 |
Filed: |
April 19, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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693018 |
May 3, 1991 |
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565544 |
Jun 29, 1990 |
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84653 |
Aug 12, 1987 |
4948734 |
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Current U.S.
Class: |
514/2.3;
536/23.71; 424/93.461 |
Current CPC
Class: |
C07K
14/325 (20130101); A01N 37/46 (20130101); A01N
63/50 (20200101); C12N 1/205 (20210501); A01N
63/50 (20200101); A01N 63/23 (20200101); C12R
2001/075 (20210501) |
Current International
Class: |
A01N
37/44 (20060101); A01N 37/46 (20060101); A01N
63/00 (20060101); A01N 63/02 (20060101); C07K
14/325 (20060101); C07K 14/195 (20060101); A01N
063/02 (); C12N 015/32 () |
Field of
Search: |
;536/23.71 ;530/350
;424/936 ;514/2 |
Other References
Coles, G. C.. (1986) "Anthelmintic Resistance in Sheep" Veterinary
Clinics of North America, Food Animal Practice 2(2):423-428. .
Ciordia, H., and W. E. Bizzell (1961) "A Preliminary Report on the
Effects of Bacillus thuringiensis var. thuringiensis Berliner on
the Development of the Free-Living Stages of Some Cattle Nematodes"
Jornal of Parasitology 47:41 *abstract*. .
Bottjer, Kurt P., Leon W. Bone, and Sarjeet S. Gill (1985)
"Nematoda: Susceptibility of the Egg to Bacillus thuringiensis
Toxins" Experimental Parasitology 60:239-244. .
Ignoffo, C. M., and V. H. Dropkin (1977) "Deleterious Effects of
the Thermostable Toxin of Bacillus thuringiensis on Species of
Soil-Inhabiting, Myceliophagus, and Plant-Parasitic Nematodes"
Journal of the Kansas Entomological Society 50(3):394-398. .
Prichard, R. K., C. A. Hall, J. D. Kelly, I. C. A. Martin, and A.
D. Donald (1980) "The Problem of Anthelmintic Resistance in
Nematodes" Australian Veterinary Journal 56:239-251..
|
Primary Examiner: Wax; Robert A.
Assistant Examiner: Hendricks; Keith D.
Attorney, Agent or Firm: Saliwanchik & Saliwanchik
Parent Case Text
CROSS-REFERENCE TO A RELATED APPLICATION
This is a division of co-pending application Ser. No. 07/693,018,
filed May 3, 1991, now abandoned, which is a continuation-in-part
of co-pending application Ser. No. 07/565,544, filed on Aug. 10,
1990, now abandoned, which is a continuation-in-part of application
Ser. No. 084,653, filed on Aug. 12, 1987, now U.S. Pat. No.
4,948,734.
Claims
I claim:
1. A process for controlling nematodes which comprises contacting
said nematodes with a nematode-controlling effective mount of a
toxin encoded by a Bacillus thuringiensis gene obtained from the
nematicidally-active Bacillus thuringiensis isolate designated B.t-
PS63B, said gone being found on a 4.4 kbp Xbal band by restriction
fragment polymorphism analysis, and comprising an N-terminal amino
acid of
QLQAQPLIPYNVLA; SEQ ID NO, 9,
and comprising internal amino add sequence of
VQRILDBKLSFQLIK; SEQ ID NO. 11,
said 4.4 kbp.multidot.XbaI band hydridizing under standard Southern
blot conditions to an approximately 460 bp fragment which is
generated by PCR using as primers SEQ ID NO. 17 and SEQ ID. 18; or
a fragment thereof sufficient to encode a nematicidally-active
toxin; wherein the nematode is selected from the group consisting
of genera Haemonchus, Trichostrongylus, Osrertagia, Nematodirus,
Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia,
Trichuris, Strongylus, Trichonema, Dictyocaulus, Capillaria,
Heterakis, Toxocara, Ascaridia, Oxyuris, Ancylogoma, Uncinaria,
Toxascaris, Caenorhabditis, Parascaris, Bursaphalenchus,
Criconenella, Ditylenchus, Globodera, Helicotylenchus, Heterodera,
Meloidogyne, Pratylenchus, Radolpholus, Rotelynchus, and
Tylenchus.
2. A process for controlling nematodes which comprises containing
said neroerodes with a nematode-controlling effective mount of a
toxin encoded by a gene obtained from the nematicidally-active
Bacillus thuringiensis isolate designated B.t PS63B, said gene
found on a 4.4 kbp XbaI band by restriction fragment polymorphism
analysis, and comprising an N-terminal amino add sequence of:
QLQAQPLIPYNVLA; SEQ ID NO. 9
and comprising an internal amino acid sequence of
VQRILDEKLSFQLIK; SEQ ID NO. 11,
said 4,4 kbp XbaI band hybridizing under standard Southern blot
conditions to approximately 460 bp fragment which is generated by
PCR using as primers SEQ ID NO. 17 and SEQ ID NO. 18; or a fragment
thereof sufficient to encode a nematicidally-active toxin.
Description
BACKGROUND OF THE INVENTION
Regular use of chemicals to control unwanted organisms can select
for drug resistant strains. This has occurred in many species of
economically important insects and has also occurred in nematodes
of sheep, goats, and horses. The development of drug resistance
necessitates a continuing search for new control agents having
different modes of action.
In recent times, the accepted methodology for control of nematodes
has centered around the drug benzimidazole and its congeners. The
use of these drugs on a wide scale has led to many instances of
resistance among nematode populations (Prichard, R. K. et al. "The
problem of anthelmintic resistance in nematodes," Austr. Vet. J.
56:239-251; Coles, G. C. [1986] "Anthelmintic resistance in sheep,"
In Veterinary Clinics of North America: Food Animal Practice, Vol
2:423-432 [Herd, R. P., eds.] W. B. Saunders, N.Y.). There are more
than 100,000 described species of nematodes.
The bacterium Bacillus thuringiensis (B.t.) produces a
.delta.-endotoxin polypeptide that has been shown to have activity
against a rapidly growing number of insect species. The earlier
observations of toxicity only against lepidopteran insects have
been expanded with descriptions of B.t. isolates with toxicity to
dipteran and coleopteran insects. These toxins are deposited as
crystalline inclusions within the organism. Many strains of B.t.
produce crystalline inclusions with no demonstrated toxicity to any
insect tested.
A small number of research articles have been published about the
effects of delta endotoxins from B. thuringiensis species on the
viability of nematode eggs. Bottjer, Bone and Gill (Experimental
Parasitology 60:239-244, 1985) have reported that B. t. kurstaki
and B. t. israelensis were toxic in vitro to eggs of the nematode
Trichostrongylus colubriformis. In addition, 28 other B.t. strains
were tested with widely variable toxicities. The most potent had
LD.sub.50 values in the nanogram range. Ignoffo and Dropkin
(Ignoffo, C. M. and Dropkin, V. H. [1977] J. Kans. Entomol. Soc.
50:394-398) have reported that the thermostable toxin from Bacillus
thuringiensis (beta exotoxin) was active against a free-living
nematode, Panagrellus redivivus (Goodey); a plant-parasitic
nematode, Meloidogyne incognita (Chitwood); and a fungus-feeding
nematode, Aphelenchus avena (Bastien). Beta exotoxin is a
generalized cytotoxic agent with little or no specificity. Also, H.
Ciordia and W. E. Bizzell (Jour. of Parasitology 47:41 [abstract]
1961) gave a preliminary report on the effects of B. thuringiensis
on some cattle nematodes.
At the present time there is a need to have more effective means to
control the many nematodes that cause considerable damage to
susceptible hosts. Advantageously, such effective means would
employ biological agents. In parent pending application Ser. No.
084,653, there are disclosed novel isolates of Bacillus
thuringiensis having activity against nematodes. We have now
isolated, unexpectedly and advantageously, genes encoding novel
nematicidal .delta.-endotoxins from the B.t. isolates PS33F2,
PS63B, PS52A1, and PS69D1. Prior to successfully completing this
invention, we could not predict with any reasonable degree of
certainty that we could isolate a gene(s) encoding a nematicidal
toxin because of the complexity of the microbial genome.
BRIEF SUMMARY OF THE INVENTION
The subject invention concerns genes or gene fragments cloned from
novel Bacillus thuringiensis isolates designated B.t. PS33F2,
PS63B, PS52A1, and PS69D1. The genes or gene fragments of the
invention encode Bacillus thuringiensis .delta.-endotoxins which
have nematicidal activity. The genes or gene fragments can be
transferred to suitable hosts via a recombinant DNA vector.
BRIEF DESCRIPTION OF THE SEQUENCES
Sequence ID 1 is the nucleotide sequence of a gene from PS33F2.
Sequence ID 2 is the amino acid sequence of the protein expressed
by the gene from PS33F2.
Sequence ID 3 is the nucleotide sequence of a gene from PS52A1.
Sequence ID 4 is the amino acid sequence of the protein expressed
by the gene from PS52A1.
Sequence ID 5 is the nucleotide sequence of a gene from PS69D1.
Sequence ID 6 is the amino acid sequence of the protein expressed
by the gene from PS69D1.
SEQ ID NO. 7 is the N-terminal amino acid sequence for PS33F2.
SEQ ID. NO. 8 is the N-terminal amino acid sequence for PS52A1.
SEQ ID. NO. 9 is the N-terminal amino acid sequence for PS63B.
SEQ ID. NO. 10 is the N-terminal amino acid sequence for
PS69D1.
SEQ ID. NO. 11 is the N-terminal amino acid sequence for
PS63B(2).
SEQ ID. NO. 12 is a probe for 33F2A.
SEQ ID. NO. 13 is a probe for 33F2B.
SEQ ID. NO. 14 is a reverse primer used for closing the PS 33F
2toxin gene.
SEQ ID. NO. 15 is an oligonucloetide probe designated 52A1-C.
SEQ ID. NO. 16 is an oligonucleotide probe designed 69D1-D.
SEQ ID. NO. 17 is a forward primer designated 63B-A.
SEQ ID. NO. 18 is a reverse primer designated 63B-INT.
DETAILED DISCLOSURE OF THE INVENTION
The novel toxin genes or gene fragments of the subject invention
were obtained from nematode-active B. thuringiensis (B.t.) isolates
designated PS33F2, PS63B, PS52A1, and PS69D1. Subcultures of the E.
coli host harboring the toxin genes of the invention were deposited
in the permanent collection of the Northern Research Laboratory,
U.S. Department of Agriculture, Peoria, Ill., USA. The accession
numbers are as follows:
______________________________________ Culture Repository No.
Deposit Date ______________________________________ B.t. isolate
PS33F2 NRRL B-18244 July 28, 1987 B.t. isolate PS63B NRRL B-18246
July 28, 1987 B.t. isolate PS52A1 NRRL B-18245 July 28, 1987 B.t.
isolate PS69D1 NRRL B-18247 July 28, 1987 E. coli NM522(pMYC 2316)
NRRL B-18785 March 15, 1991 E. coli NM522(pMYC 2321) NRRL B-18770
February 14, 1991 E. coli NM522(pMYC 2317) NRRL B-18816 April 24,
1991 ______________________________________
The subject cultures have been deposited under conditions that
assure that access to the cultures will be available during the
pendency of this patent application to one determined by the
Commissioner of Patents and Trademarks to be entitled thereto under
37 CFR 1.14 and 35 USC 122. The deposits are available as required
by foreign patent laws in countries wherein counterparts of the
subject application, or its progeny, are filed. However, it should
be understood that the availability of a deposit does not
constitute a license to practice the subject invention in
derogation of patent rights granted by governmental action.
Further, the subject culture deposits will be stored and made
available to the public in accord with the provisions of the
Budapest Treaty for the Deposit of Microorganisms, i.e., they will
be stored with all the care necessary to keep them viable and
uncontaminated for a period of at least five years after the most
recent request for the furnishing of a sample of the deposit, and
in any case, for a period of at least 30 (thirty) years after the
date of deposit or for the enforceable life of any patent which may
issue disclosing the cultures. The depositor acknowledges the duty
to replace the deposits should the depository be unable to furnish
a sample when requested, due to the condition of the deposit(s).
All restrictions on the availability to the public of the subject
culture deposits will be irrevocably removed upon the granting of a
patent disclosing them.
The novel B.t. genes or gene fragments of the invention encode
toxins which show activity against tested nematodes. The group of
diseases described generally as helminthiasis is due to infection
of an animal host with parasitic worms known as helminths.
Helminthiasis is a prevalent and serious economic problem in
domesticated animals such as swine, sheep, horses, cattle, goats,
dogs, cats and poultry. Among the helminths, the group of worms
described as nematodes causes wide-spread and often times serious
infection in various species of animals. The most common genera of
nematodes infecting the animals referred to above are Haemonchus,
Trichostrongylus, Ostertagia, Nematodirus, Cooperia, Ascaris,
Bunostomum, Oesophagostomum, Chabertia, Trichuris, Strongylus,
Trichonema, Dictyocaulus, Capillaria, Heterakis, Toxocara,
Ascaridia, Oxyuris, Ancylostoma, Uncinaria, Toxascaris,
Caenorhabditis and Parascaris. Certain of these, such as
Nematodirus, Cooperia, and Oesophagostomum, attack primarily the
intestinal tract, while others, such as Dictyocaulus are found in
the lungs. Still other parasites may be located in other tissues
and organs of the body.
The toxins encoded by the novel B.t. genes of the invention are
useful as nematocides for the control of soil nematodes and plant
parasites selected from the genera Bursaphalenchus, Criconemella,
Ditylenchus, Globodera, Helicotylenchus, Heterodera, Melodoigyne,
Pratylenchus, Radolpholus, Rotelynchus, or Tylenchus.
Alternatively, because some plant parasitic nematodes are obligate
parasites, genes coding for nematocidal B.t. toxins can be
engineered into plant cells to yield nematode-resistant plants. The
methodology for engineering plant cells is well established (cf.
Nester, E. W., Gordon, M. P., Amasino, R. M. and Yanofsky, M. F.,
Ann. Rev. Plant Physiol. 35:387-399, 1984).
The B.t. toxins of the invention can be administered orally in a
unit dosage form such as a capsule, bolus or tablet, or as a liquid
drench when used as an anthelmintic in mammals, and in the soil to
control plant nematodes. The drench is normally a solution,
suspension or dispersion of the active ingredient, usually in
water, together with a suspending agent such as bentonitc and a
wetting agent or like excipient. Generally, the drenches also
contain an antifoaming agent. Drench formulations generally contain
from about 0.001 to 0.5% by weight of the active compound.
Preferred drench formulations may contain from 0.01 to 0.1% by
weight, the capsules and boluses comprise the active ingredient
admixed with a carrier vehicle such as starch, talc, magnesium
stearate, or dicalcium phosphate.
Where it is desired to administer the toxin compounds in a dry,
solid unit dosage form, capsules, boluses or tablets containing the
desired amount of active compound usually are employed. These
dosage forms are prepared by intimately and uniformly mixing the
active ingredient with suitable finely divided diluents, fillers,
disintegrating agents and/or binders such as starch, lactose, talc,
magnesium stearate, vegetable gums and the like. Such unit dosage
formulations may be varied widely with respect to their total
weight and content of the antiparasitic agent, depending upon the
factors such as the type of host animal to be treated, the severity
and type of infection and the weight of the host.
When the active compound is to be administered via an animal
feedstuff, it is intimately dispersed in the feed or used as a top
dressing or in the form of pellets which may then be added to the
finished feed or, optionally, fed separately. Alternatively, the
antiparasitic compounds may be administered to animals
parenterally, for example, by intraruminal, intramuscular,
intratracheal, or subcutaneous injection, in which event the active
ingredient is dissolved or dispersed in a liquid carrier vehicle.
For parenteral administration, the active material is suitably
admixed with an acceptable vehicle, preferably of the vegetable oil
variety, such as peanut oil, cotton seed off and the like. Other
parenteral vehicles, such as organic preparations using solketal,
glycerol, formal and aqueous parenteral formulations, are also
used. The active compound or compounds are dissolved or suspended
in the parenteral formulation for administration; such formulations
generally contain from 0.005 to 5% by weight of the active
compound.
When the toxins are administered as a component of the feed of the
animals, or dissolved or suspended in the drinking water,
compositions are provided in which the active compound or compounds
are intimately dispersed in an inert carrier or diluent. By inert
carrier is meant one that will not react with the antiparasitic
agent and one that may be administered safely to animals.
Preferably, a carrier for feed administration is one that is, or
may be, an ingredient of the animal ration.
Suitable compositions include feed premixes or supplements in which
the active ingredient is present in relatively large amounts and
which are suitable for direct feeding to the animal or for addition
to the feed either directly or after an intermediate dilution or
blending step. Typical carriers or diluents suitable for such
compositions include, for example, distillers' dried grains, corn
meal, citrus meal, fermentation residues, ground oyster shells,
wheat shorts, molasses solubles, corn cob meal, edible bean mill
feed, soya grits, crushed limestone and the like.
The toxin genes or gene fragments of the subject invention can be
introduced into a wide variety of microbial hosts. Expression of
the toxin gene results, directly or indirectly, in the
intracellular production and maintenance of the nematicide. With
suitable hosts, e.g., Pseudomonas, the microbes can be applied to
the situs of nematodes where they will proliferate and be ingested
by the nematodes. The result is a control of the nematodes.
Alternatively, the microbe hosting the toxin gene can be treated
under conditions that prolong the activity of the toxin produced in
the cell. The treated cell then can be applied to the environment
of target pest(s). The resulting product retains the toxicity of
the B.t. toxin.
Where the B.t. toxin gene or gene fragment is introduced via a
suitable vector into a microbial host, and said host is applied to
the environment in a living state, it is essential that certain
host microbes be used. Microorganism hosts are selected which are
known to occupy the "phytosphere" (phylloplane, phyllosphere,
rhizosphere, and/or rhizoplane) of one or more crops of interest.
These microorganisms are selected so as to be capable of
successfully competing in the particular environment (crop and
other insect habitats) with the wild-type microorganisms, provide
for stable maintenance and expression of the gene expressing the
polypeptide pesticide, and, desirably, provide for improved
protection of the nematicide from environmental degradation and
inactivation.
A large number of microorganisms are known to inhabit the
phylloplane (the surface of the plant leaves) and/or the riosphere
(the soft surrounding plant roots) of a wide variety of important
crops. These microorganisms include bacteria, algae, and fungi. Of
particular interest are microorganisms, such as bacteria, e.g.,
genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas,
Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius,
Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter,
Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly
yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces,
Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular
interest are such phytosphere bacterial species as Pseudomonas
syringae. Pseudomonas fluorescens, Serratia marcescens, Acetobacter
xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides,
Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus,
and Azotobacter vinlandii; and phytosphere yeast species such as
Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,
Cryptococcus albidus, C. diffiuens, C. laurentii, Saccharomyces
rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S.
odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of
particular interest are the pigmented microorganisms.
A wide variety of ways are known and available for introducing the
B.t. genes or gene fragments expressing the toxin into the
microorganism host under conditions which allow for stable
maintenance and expression of the gene. The transformants can be
isolated in accordance with conventional ways, usually employing a
selection technique, which allows for selection of the desired
organism as against unmodified organisms or transferring organisms,
when present. The transformants then can be tested for nematicidal
activity.
Suitable host cells, where the nematicide-containing cells will be
treated to prolong the activity of the toxin in the cell when the
then treated cell is applied to the environment of target pest(s),
may include either prokaryotes or eukaryotes, normally being
limited to those cells which do not produce substances toxic to
higher organisms, such as mammals. However, organisms which produce
substances toxic to higher organisms could be used, where the toxin
is unstable or the level of application sufficiently low as to
avoid any possibility of toxicity to a mammalian host. As hosts, of
particular interest will be the prokaryotes and the lower
eukaryotes, such as fungi. Illustrative prokaryotes, both
Gram-negative and positive, include Enterobacteriaceae, such as
Escherichia, Erwinia, Shigella, Salmonella, and Proteus;
Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as
photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,
Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such
as Pseudomonas and Acetobacter; Azotobacteraceae and
Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes
and Ascomycetes, which includes yeast, such as Saccharomyces and
Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,
Aureobasidium, Sporobolomyces, and the like.
Characteristics of particular interest in selecting a host cell for
purposes of production include ease of introducing the B.t. gene or
gene fragment into the host, availability of expression systems,
efficiency of expression, stability of the nematicide in the host,
and the presence of auxiliary genetic capabilities. Characteristics
of interest for use as a nematicide microcapsule include protective
qualities for the nematicide, such as thick cell walls,
pigmentation, and intracellular packaging or formation of inclusion
bodies; leaf affinity; lack of mammalian toxicity; attractiveness
to pests for ingestion; ease of killing and fixing without damage
to the toxin; and the like. Other considerations include ease of
formulation and handling, economics, storage stability, and the
like.
Host organisms of particular interest include yeast, such as
Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and
Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp.,
Erwinia sp. and Flavobacterium sp.; or such other organisms as
Escherichia, Lactobacillus sp., Bacillus sp., and the like.
Specific organisms include Pseudomonas aeruginosa, Pseudomonas
fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis,
Escherichia coli, Bacillus subtilis, and the like.
The cell will usually be intact and be substantially in the
proliferative form when treated, rather than in a spore form,
although in some instances spores may be employed.
Treatment of the microbial cell, e.g., a microbe containing the
B.t. toxin gene or gene fragment, can be by chemical or physical
means, or by a combination of chemical and/or physical means, so
long as the technique does not deleteriously affect the properties
of the toxin, nor diminish the cellular capability in protecting
the toxin. Examples of chemical reagents are halogenating agents,
particularly halogens of atomic no. 17-80. More particularly,
iodine can be used under mild conditions and for sufficient time to
achieve the desired results. Other suitable techniques include
treatment with aldehydes, such as formaldehyde and glutaraldehyde;
anti-infectives, such as zephiran chloride and cetylpyridinium
chloride; alcohols, such as isopropyl and ethanol; various
histologic fixatives, such as Bouin's fixative and Helly's fixative
(See: Humason, Gretchen L., Animal Tissue Techniques, W. H. Freeman
and Company, 1967); or a combination of physical (heat) and
chemical agents that preserve and prolong the activity of the toxin
produced in the cell when the cell is administered to the host
animal. Examples of physical means are short wavelength radiation
such as gamma-radiation and X-radiation, freezing, UV irradiation,
lyophilization, and the like.
The cells generally will have enhanced structural stability which
will enhance resistance to environmental conditions. Where the
pesticide is in a proform, the method of inactivation should be
selected so as not to inhibit processing of the proform to the
mature form of the pesticide by the target pest pathogen. For
example, formaldehyde will crosslink proteins and could inhibit
processing of the proform of a polypeptide pesticide. The method of
inactivation or killing retains at least a substantial portion of
the bio-availability or bioactivity of the toxin.
The cellular host containing the B.t. nematicidal gene or gene
fragment may be grown in any convenient nutrient medium, where the
DNA construct provides a selective advantage, providing for a
selective medium so that substantially all or all of the cells
retain the B.t. gene or gene fragment. These cells may then be
harvested in accordance with conventional ways. Alternatively, the
cells can be treated prior to harvesting.
The B.t. cells may be formulated in a variety of ways. They may be
employed as wettable powders, granules or dusts, by mixing with
various inert materials, such as inorganic minerals
(phyllosilicates, carbonates, sulfates, phosphates, and the like)
or botanical materials (powdered corncobs, rice hulls, walnut
shells, and the like). The formulations may include
spreader-sticker adjuvants, stabilizing agents, other pesticidal
additives, or surfactants. Liquid formulations may be aqueous-based
or non-aqueous and employed as foams, gels, suspensions,
emulsifiable concentrates, or the like. The ingredients may include
rheological agents, surfactants, emulsifiers, dispersants, or
polymers.
The nematicide concentration will vary widely depending upon the
nature of the particular formulation, particularly whether it is a
concentrate or to be used directly. The nematicide will be present
in at least 1% by weight and may be 100% by weight. The dry
formulations will have from about 1-95% by weight of the nematicide
while the liquid formulations will generally be from about 1-60% by
weight of the solids in the liquid phase. The formulations will
generally have from about 10.sup.2 to about 10.sup.4 cells/mg.
These formulations will be administered at about 50 mg (liquid or
dry) to 1 kg or more per hectare.
The formulations can be applied to the environment of the
nematodes, e.g., plants, soil or water, by spraying, dusting,
sprinkling, or the like.
Following are examples which illustrate procedures, including the
best mode, for practicing the invention. These examples should not
be construed as limiting. All percentages are by weight and all
solvent mixture proportions are by volume unless otherwise
noted.
Example 1--Culturing B.t. Isolates of the Invention
A subculture of a B.t. isolate can be used to inoculate the
following medium, a peptone, glucose, salts medium.
______________________________________ Bacto Peptone 7.5 g/l
Glucose 1.0 g/l KH.sub.2 PO.sub.4 3.4 g/l K.sub.2 HPO.sub.4 4.35
g/l Salts Solution 5.0 ml/l CaCl.sub.2 Solution 5.0 ml/l Salts
Solution (100 ml) MgSO.sub.4.7H.sub.2 O 2.46 g MnSO.sub.4.H.sub.2 O
0.04 g ZnSO.sub.4.7H.sub.2 O 0.28 g FeSO.sub.4.7H.sub.2 O 0.40 g
CaCl.sub.2 Solution (100 ml) CaCl.sub.2.2H.sub.2 O 3.66 g pH 7.2
______________________________________
The salts solution and CaCl.sub.2 solution are filter-sterilized
and added to the autoclaved and cooked broth at the time of
inoculation. Flasks are incubated at 30.degree. C. on a rotary
shaker at 200 rpm for 64 hr.
Example 2--Purification of Protein and Amino Acid Sequencing
The B.t. isolates PS33F2, PS63B, PS52A1, and PS69D1 were cultured
as described in Example 1. The parasporal inclusion bodies were
partially purified by sodium bromide (28-38%) isopycnic gradient
centrifugation (Pfannenstiel, M. A., E. J. Ross, V. C. Kramer, and
K. W. Nickerson [1984] FEMS Microbiol. Lett. 21:39). The proteins
toxic for the nematode Caenorhabditis elegans were bound to PVDF
membranes (Millipore, Bedford, MA) by western blotting techniques
(Towbin, H., T. Staehlelin, and K. Gordon [1979] Proc. Natl. Atari.
Sci. USA 76:4350) and the N-terminal amino acid sequences were
determined by the standard Edman reaction with an automated
gas-phase sequenator (Hunkapiller, M. W., R. M. Hewick, W. L.
Dreyer, and L. E. Hood[1983] Meth. Enzymol. 91:399). The sequences
obtained were:
PS33F2ATLNEVYPVN
PS52A1 MIIDSKTTLPRHSLINT
PS63B QLQAQPLIPYNVLA
PS69D1 MILGNGKTLPKHIRLAHIFATQNS
In addition, internal amino acid sequence data were derived for
PS63B. The toxin protein was partially digested with Staphylococcus
aureus V8 protease (Sigma Chem. Co., St. Louis, MO) essentially as
described (Cleveland, D. W., S. G. Fischer, M. W. Kirsclmer, and U.
K. Laemrnli [1977] J. Biol. Chem. 252:1102). The digested material
was blotted onto PVDF membrane and a ca. 28 kDa limit peptide was
selected for N-terminal sequencing as described above. The sequence
obtained was:
63B(2) VQRILDEKLSFQLIK
From these sequence data oligonucleotide probes were designed by
utilizing a codon frequency table assembled from available sequence
data of other B.t. toxin genes. The probes were synthesized on an
Applied Biosystems, Inc. DNA synthesis machine.
Protein purification and subsequent amino acid analysis of the
N-terminal peptides listed above has led to the deduction of
several oligonucleotide probes for the isolation of toxin genes
from nematicidal B.t. isolates. RFLP analysis of restricted total
cellular DNA using radiolabeled oligonucleotide probes has
elucidated different genes or gene fragments.
Example 3--Cloning of a Novel Toxin Gene From B.t. PS33F2 and
Transformation into Escherichia coli
Total cellular DNA was prepared from B.t. PS33F2 cells grown to an
optical density, at 600 nm, of 1.0. Cells were pelleted by
centrifugation and resuspended in protoplast buffer (20 mg/ml
lysozyme in 0.3M sucrose, 25 mM Tris-Cl [pH 8.0], 25 mM EDTA).
After incubation at 37.degree. C. for 1 h, protoplasts were lysed
by the addition of nine volumes of a solution of 0.1M NaCl, 0.1%
SDS, 0.1 M Tris-C1 followed by two cycles of freezing and thawing.
The cleared lysate was extracted twice with phenol:chloroform
(1:1). Nucleic acids were precipitated with two volumes of ethanol
and pelleted by centrifugation. The pellet was resuspended in 10mM
Tris-Cl, 1 mM EDTA (TE) and RNase was added to a final
concentration of 50 .mu.g/ml. After incubation at 37.degree. C. for
1 h, the solution was extracted once each with phenol:chloroform
(1:1) and TE-saturated chloroform. DNA was precipitated from the
aqueous phase by the addition of one-tenth volume of 3M NaOAc and
two volumes of ethanol. DNA was pelleted by centrifugation, washed
with 70% ethanol, dried, and resuspended in TE.
Plasmid DNA was extracted from protoplasts prepared as described
above. Protoplasts were lysed by the addition of nine volumns of a
solution of 10 mM Tris-Cl, 1 mM EDTA, 0.085 N NaOH, 0.1% SDS,
pH=8.0. SDS was added to 1% final concentration to complete lysis.
One-half volume of 3M KOAc was then added and the cellular material
was precipitated overnight at 4.degree. C. After centrifugation,
the DNA was precipitated with ethanol and plasmids were purified by
isopycnic centrifugation on cesium chloride-ethldium bromide
gradients.
Restriction Fragment Length Polymorphism (RFLP) analyses were
performed by standard hybridization of Southern blots of PS33F2
plasmid and total cellular DNA with 32P-labelled oligonucleotide
probes designed to the N-terminal amino acid sequence disclosed in
Example 2.
Probe 33F2A: 5'GCA/F ACA/T TYA AAT GAA GTA/T TAT 3'
Probe 33F2B: 5'AAT GAA GTA/T TAT CCA/T GTA/T AAT 3'
Hybridizing bands included an approximately 5.85 kbp EcoRI
fragment. Probe 33F2A and a reverse PCR primer were used to amplify
a DNA fragment of approximately 1.8 kbp for use as a hybridization
probe for cloning the PS33F2 toxin gene. The sequence of the
reverse primer was:
5'GCAAGCGGCCGCTTATGGAATAAATTCAATT G A/G TC T/A A 3'
A gene library was constructed from PS33F2 plasmid DNA digested
with EcoRI. Restriction digests were fractionated by agarose gel
electrophoresis. DNA fragments 4.3-6.6 kbp were excised from the
gel, electroeluted from the gel slice, and recovered by ethanol
precipitation after purification on an Elutip-D ion exchange column
(Schleicher and Schuel, Keene NH). The EcoRI inserts were ligated
into EcoRI-digested pHTBluelI (an E. coli./B. thuringiensis shuttle
vector comprised of pBluescript S/K [Stratagene] and the
replication origin from a resident B.t. plasmid [D. Lereclus et al.
1989. FEMS Microbial. Lett. 60:211-218]). The ligation mixture was
transformed into frozen, competent NM522 cells (ATCC 47000).
Transformants were plated on LB agar containing ampicillin,
isopropyl -(Beta)-D-thiogalactoside (IPTG), and
5-bromo-4-chloro-3-indolyl-(Beta)-D-galactoside (XGAL). Colonies
were screened by hybridization with the radiolabeled PCR amplified
probe described above. Plasmids were purified from putative toxin
gene clones by alkaline lysis and analyzed by agarose gel
electrophoresis of restriction digests. The desired plasmid
construct, pMYC2316, contains an approximately 5.85 kbp EcoRI
insert; the toxin gene residing on this DNA fragment (33F2a) is
novel compared to the DNA sequences of other toxin genes encoding
nematicidal proteins.
Plasmid pMYC2316 was introduced into the acrystallfferous (Cry-)
B.t. host, HD-1 CryB (A. Aronson, Purdue University, West
Lafayette, IN) by electroporation. Expression of an approximately
120-140 kDa crystal protein was verified by SDS-PAGE analysis.
Crystals were purified on NaBr gradients (M. A. Pfannenstiel et al.
1984. FEMS Microbiol. Lett. 21:39) for determination of toxicity of
the cloned gene product to Pratylenchus spp.
Example 4--Activity of the B.t. Gene Product PS33F2 Against the
Plant Nematode Pratylenchus spp.
Pratylenchus spp. was reared aseptically on excised corn roots in
Gamburg's B5 medium (GIBCO.RTM. Laboratories, Grand Island, N.Y.)
Bioassays were done in 24 well assay plates (Corning #25820) using
L 3-4 larvae as described by Tsai and van Gundy (J. Nematol.
22(3):327-332). Approximately 20 nematodes were placed in each
well. A total of 80-160 nematodes were used in each treatment.
Samples of protein were suspended in an aqueous solution using a
hand-held Dounce homogenizer.
Mortality was assessed visually 3 days after treatment. Larvae that
were nearly straight and not moving were considered moribund.
Representative results are as follows:
______________________________________ PS33F2a (ppm) % Moribund
______________________________________ 0 12 75 78
______________________________________
Species of Pratylenchus, for example P. scribneri, are known
pathogens of many economically important crops including corn,
peanuts, soybean, alfalfa, beans, tomato, and citrus. These "root
lesion" nematodes are the second most economically damaging genus
of plant parasitic nematodes (after Meloidogyne--the "root knot"
nematode), and typify the migratory endoparasites.
Example 5--Molecular Cloning of Gene Encoding a Novel Toxin From
Bacillus thuringiensis strain PS52A1
Total cellular DNA was prepared from Bacillus thuringiensis PS52A1
(B.t. PS52A1) as disclosed in Example 3.
RFLP analyses were performed by standard hybridization of Southern
blots of PS52A1 DNA with a .sup.32 P-labeled oligonucleotide probe
designed from the N-terminal amino acid sequence disclosed in
Example 2. The sequence of this probe is:
5'ATG ATY ATT GAT TCT AAA ACA ACA TTA CCA AGA CAT TCA/T TYA ATA/T
AAT ACA/T ATA/T AA 3'
This probe was designated 52A1-C. Hybridizing bands included an
approximately 3.6 kbp Hind-III fragment and an approximately 8.6
kbp EcoRV fragment. A gene library was constructed from PS52A1 DNA
partially digested with Sau3A. Partial restriction digests were
fractionated by agarose gel electrophoresis. DNA fragments 6.6 to
23 kbp in size were excised from the gel, electroeluted from the
gel slice, and recovered by ethanol precipitation after
purification on an Elutip-D ion exchange column. The Sau3A inserts
were ligated into BamHI-digested LambdaGem-11 (Promega).
Recombinant phage were packaged and plated on E. coli KW25 1 cells
(Promega). Plaques were screened by hybridization with the
radiolabeled 52A1-C oligonucleotide probe disclosed above.
Hybridizing phage were plaque-purified and used to infect liquid
cultures of E. coli KW25 1 cells for isolation of phage DNA by
standard procedures (Maniatis et al.). For subcloning, preparative
amounts of DNA were digested with EcoRI and SalI, and
electrophoresed on an agarose gel. The approximately 3.1 kbp band
containing the toxin gene was excised from the gel, electroeluted
from the gel slice, and purified by ion exchange chromatography as
above. The purified DNA insert was ligated into EcoRI+Sal-digested
pHTBlueII (an E. coli/B. thuringiensis shuttle vector comprised of
pBluescript S/K [Stratagene] and the replication origin from a
resident B.t. plasmid [D. Lereclus et al. 1989. FEMS Microbiology
Letters 60:211-218]). The ligation mix was used to transform
frozen, competent E. coli NM522 cells (ATTCC 47000). Transformants
were plated on LB agar containing ampicillin,
isoprypyl-(Beta)-D-thiogalactoside (IPTG), and
5-Bromo-4-Chloro-3-indolyl-(Beta)-D-galactoside (XGAL). Plasmids
were purified from putative recombinants by alkaline lysis
(Maniatis et al.) and analyzed by electrophoresis of EcoRI and SalI
digests on agarose gels. The desired plasmid construct, pMYC2321
contains a tom gene that is novel compared to the maps of other
toxin genes encoding nematicidal proteins.
Plasmid pMYC2321 was introduced into an acrystalliferous (Cry-)
B.t. host by electroporation. Expression of an approximately 55-60
kDa crystal protein was verified by SDS-PAGE analysis.
NaBr-purified crystals were prepared as described in Example 3 for
determination of toxicity of the cloned gene product to
Pratylenchus spp.
Example 6--Activity of the B.t. PS52A1 Toxin Protein and Gene
Product Against the Root Lesion Nematode, Pratylenchus
scribneri
Pratylenchus scribneri was reared aseptically on excised corn roots
in Gamburg's B5 medium (GIBCO.RTM. Laboratories, Grand Island,
N.Y.). Bioassays were done in 24 well assay plates (Corning #25820)
using L 3-4 larvae as described by Tsai and Van Gundy (J. Nematol.
22(3):327-332). Approximately 20 nematodes were placed in each
well. A total of 80-160 nematodes were used in each treatment.
Samples of protein were suspended in aqueous solution using a
hand-held homogenizer.
Mortality was assessed by prodding with a dull probe 7 days after
treatment. Larvae that did not respond to prodding were considered
moribund. Representative results are shown below.
______________________________________ Rate Percent (ppm) Moribund
______________________________________ 200 75 Control 5
______________________________________
Example 7--Molecular Cloning of Gene Encoding a Novel Toxin From
Bacillus Thuringiensis strain PS 69D1
Total cellular DNA was prepared from PS69D1 (B.t. PS69D1) as
disclosed in Example 3. RFLP analyses were performed by standard
hybridization of Southern blots of PS69D1 DNA with a 32P-labeled
oligonucleotide probe designated as 69D1-D. The sequence of the
69D1-D probe was:
5'AAA CAT ATF AGA TTA GCA CAT ATF TTF GCA ACA CAA AA 3'Hybridizing
bands included an approximately 2.0 kbp HindIII fragment.
A gene library was constructed from PS69D1 DNA partially digested
with Sau3A. Partial restriction digests were fractionated by
agarose gel electrophoresis. DNA fragments 6.6 to 23 kbp in size
were excised from the gel, electroeluted from the gel slice, and
recovered by ethanol precipitation after purification on an
Elutip-D ion exchange column. The Sau3A inserts were ligated into
BamHI-digested LambdaGem-11 (Promega, Madison, WI). Recombinant
phage were packaged and plated on E. coli KW25 1 cells (Promega,
Madison, WI). Plaques were screened by hybridization with the
radiolabeled 69D1-D oligonucleotide probe. Hybridizing phage were
plaque-purified and used to infect liquid cultures of E. coli KW251
cells for isolation of phage DNA by standard procedures (Maniatis
et al. [1982] Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor, N.Y.). For subcloning, preparative amounts of DNA were
digested with HindIII and electrophoresed on an agarose gel. The
approximately, 2.0 kbp band containing the toxin gene was excised
from the gel, electroeluted from the gel slice, and purified by ion
exchange chromatography as above. The purified DNA insert was
ligated into HindIII-digested pHTBlueII (and E. coli/B.t. shuttle
vector comprised of pBluescript S/K (Stratagene, San Diego, CA) and
the replication origin from a resident B.t. plasmid (D. Lereclus et
al [1989] FEMS Microbiol. Lett. 60:211-218)). The ligation mix was
used to transform frozen, competent E. coli NM522 cells (ATCC
47000). Transformants were plated on LB agar contianging
5-Bromo-4-Chloro-3-indolyl-(Beta)-D-galactoside (XGAL). Plasmids
were purified from putative recombinants by alkaline lysis
(Maniatis et al., ibid.) and analyzed by electorphoresis of HindIII
digests on agarose gels. The desired plasmid construct, pMYC2317,
contains a toxin gene that is novel compared to the maps of other
toxin genes encoding insecticidal proteins.
Example 8-Molecular Cloning of a Gene Encoding a Novel Toxin from
Bacillus thuringiensis Strain PS63B
Example 2 shows the aminoterminal and internal polypeptide
sequences of the PS63B toxin protein as determined by standard
Edman protein sequencing. From these sequences, two oligonucleotide
primers were designed using a codon frequency table assembled from
B.t. genes encoding .delta.-endotoxins. The sequence of the forward
primer (63B-A) was complementary to the predicted DNA sequence at
the 5'end of the gene:
63B-A-5'CAA T/CTA CAA GCA/T CAA CC 3'
The sequence of the reverse primer (63B-INT) was complementary to
the inverse of the internal predicted DNA sequence:
63B-INT -5'TTC ATC TAA AAT TCT TTG AJTAC 3'
These primers were used in standard polymerase chain reactions
(Cetus Corporation) to amplify an approximately 460 bp fragment of
the 63B toxin gene for use as a DNA cloning probe. Standard
Southern blots of total cellular DNA from PS63B were hybridized
with the radiolabeled PCR probe. Hybridizing bands included an
approximately 4.4 kbp XbaI fragment, an approximately 2.0 kbp
HindlII fragment, and an approximately 6.4 kbp SpeI fragment.
Example 9--Insertion of Toxin Gene Into Plants
The novel gene coding for the novel nematicidal toxin, as disclosed
herein, can be inserted into plant cells using the Ti plasmid from
Agrobacter tumefaciens. Plant cells can then be caused to
regenerate into plants (Zambryski, P., Joos, H., Gentello, C.,
Leeroans, J., Van Montague, M. and Schell, J [1983]Cell
32:1033-1043). A particularly useful vector in this regard is
pEND4k (Klee, H. J., Yanofsky, M. F. and Nester, E. W. [1985]
Biofrechnology 3:637-642). This plasmid can replicate both in plant
cells and in bacteria and has multiple cloning sites for passenger
genes. The toxin gene, for example, can be inserted into the BamHI
site of pEND4K, propagated in E. coli, and transformed into
appropriate plant cells.
Example 10--Cloning of Novel Hybrid B. thuringiensis Genes Into
Baculoviruses
The novel hybrid gene of the invention can be cloned into
baculoviruses such as Autographa californica nuclear polyhedrosis
virus (AcNPV). Plasmids can be constructed that contain the AcNPV
genome cloned into a commercial cloning vector such as pUC8. The
AcNPV genome is modified so that the coding region of the
polyhedrin gene is removed and a unique cloning site for a
passenger gene is placed directly behind the polyhedrin promoter.
Examples of such vectors are pGP-B6874, described by Pennock et al.
(Pennook, G.d., Shoemaker, C. and Miller, L. K. [1984] Mol. Cell.
Biol. 4:399-406), and pAC380, described by Smith et al. (Smith, G.
E., Summers, M. D. and Fraser, M. J. [1983] Mol Cell. Biol.
3:2156-2165). The gene coding for the novel protein toxin of the
invention can be modified with BamHI linkers at appropriate regions
both upstream and downstream from the coding region and inserted
into the passenger site of one of the AcNPV vectors.
It is well known in the art that the amino acid sequence of a
protein is determined by the nucleotide sequence of the DNA.
Because of the redundancy of the genetic code, i.e., more than one
coding nucleotide triplet (codon) can be used for most of the amino
acids used to make proteins, different nucleotide sequences can
code for a particular amino acid. Thus, the genetic code can be
depicted as follows:
______________________________________ Phenylalanine (Phe) TTK
Histidine (His) CAK Leucine (Leu) XTY Glutamine (Gln) CAJ
Isoleucine (Ile) ATM Asparagine (Asn) AAK Methionine (Met) ATG
Lysine (Lys) AAJ Valine (Val) GTL Aspartic acid (Asp) GAK Serine
(Ser) QRS Glutamic acid (Glu) GAJ Proline (Pro) CCL Cysteine (Cys)
TGK Threonine (Thr) ACL Tryptophan (Trp) TGG Alanine (Ala) GCL
Arginine (Arg) WGZ Tyrosine (Tyr) TAK Glycine (Gly) GGL Termination
signal TAJ ______________________________________
Key: Each 3-letter deoxynucleotide triplet corresponds to a
trinucleotide of mRNA, having a 5'-end on the left and a Y-end on
the right. All DNA sequences given herein are those of the strand
whose sequence correspond to the mRNA sequence, with thymine
substituted for uracil. The letters stand for the purine or
pyrimidine bases forming the deoxynucleotide sequence.
A=adenine
G=guanine
C=cytosine
T=thymine
X=T or C if Y is A or G
X=C if Y is C or T
Y=A, G, C or T if X is C
Y=A or G if X is T
W=C or A if Z is A or G
W--C if Z is C or T
Z=A, G, Cor T if W is C
Z=A or G if W is A
QR=TC if S is A, G, C or T; alternatively
QR=AG ff S is T or C
J=AorG
K=TorC
L=A, T, CorG
M=A, CorT
The above shows that the novel amino acid sequence of the B.t.
toxins can be prepared by equivalent nucleotide sequences encoding
the same amino acid sequence of the protein. Accordingly, the
subject invention includes such equivalent nucleotide sequences. In
addition it has been shown that proteins of identified structure
and function may be constructed by changing the amino acid sequence
if such changes do not alter the protein secondary structure
(Kaiser, E. T. and Kezdy, F. J. [1984] Science 223:249-255). Thus,
the subject invention includes mutants of the amino acid sequence
depicted herein which do not alter the protein secondary structure,
or if the structure is altered, the biological activity is
substantially retained. Further, the invention also includes
mutants of organisms hosting all or pan of a toxin encoding a gene
of the invention. Such microbial mutants can be made by techniques
well known to persons skilled in the art. For example, UV
irradiation can be used to prepare mutants of host organisms.
Likewise, such mutants may include asporogenous host cells which
also can be prepared by procedures well known in the art.
The various methods employed in the preparation of the plasmids and
transformation of host organisms are well known in the art. These
procedures are all described in Maniatis, T., Fritsch, E. F., and
Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, N.Y. Thus, it is within the skill of
those in the genetic engineering art to extract DNA from microbial
cells, perform restriction enzyme digestions, electrophorese DNA
fragments, tail and anneal plasmid and insert DNA, ligate DNA,
transform cells, prepare plasmid DNA, electrophorese proteins, and
sequence DNA.
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 18 (2) INFORMATION FOR SEQ ID NO:1 (PS33F2): (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 3771 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus
thuringiensis (C) INDIVIDUAL ISOLATE: 33f2 (vii) IMMEDIATE SOURCE:
(B) CLONE: 33f2a (ix) FEATURE: (A) NAME/KEY: miscfeature (B)
LOCATION: 4..24 (D) OTHER INFORMATION: /function="oligonucleotide
hybridization probe" /product="GCA/T ACA/T TTA AAT GAA GTA/T TAT"
/standardname="probe a" /note="Probe A" (ix) FEATURE: (A) NAME/KEY:
miscfeature (B) LOCATION: 13..33 (D) OTHER INFORMATION:
/function="oligonucleotide hybridization probe" /product="AAT GAA
GTA/T TAT CCA/T GTA/T AAT" /standardname="Probe B" /label=probe-b
/note="probe b" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATGGCTACACTTAATGAAGTATATCCTGTGAATTATAATGTATTATCTTCTGATGCTTTT60
CAACAATTAGATACAACAGGTTTTAAAAGTAAATATGATGAAATGATAAAAGCATTCGAA120
AAAAAATGGA AAAAAGGGGCAAAAGGAAAAGACCTTTTAGATGTTGCATGGACTTATATA180
ACTACAGGAGAAATTGACCCTTTAAATGTAATTAAAGGTGTTTTATCTGTATTAACTTTA240
ATTCCTGAAGTTGGTACTGTGGCCTCTGCAGCAAGTACTATTGTAAGTTTTATTTG GCCT300
AAAATATTTGGAGATAAACCAAATGCAAAAAATATATTTGAAGAGCTCAAGCCTCAAATT360
GAAGCATTAATTCAACAAGATATAACAAACTATCAAGATGCAATTAATCAAAAAAAATTT420
GACAGTCTTCAGAAAACAATTAATCTATATACA GTAGCTATAGATAACAATGATTACGTA480
ACAGCAAAAACGCAACTCGAAAATCTAAATTCTATACTTACCTCAGATATCTCCATATTT540
ATTCCAGAAGGATATGAAACTGGAGGTTTACCTTATTATGCTATGGTTGCTAATGCTCAT600
ATATTATTGT TAAGAGACGCTATAGTTAATGCAGAGAAATTAGGCTTTAGTGATAAAGAA660
GTAGACACACATAAAAAATATATCAAAATGACAATACACAATCATACTGAAGCAGTAATA720
AAAGCATTCTTAAATGGACTTGACAAATTTAAGAGTTTAGATGTAAATAGCTATAA TAAA780
AAAGCAAATTATATTAAAGGTATGACAGAAATGGTTCTTGATCTAGTTGCTCTATGGCCA840
ACTTTCGATCCAGATCATTATCAAAAAGAAGTAGAAATTGAATTTACAAGAACTATTTCT900
TCTCCAATTTACCAACCTGTACCTAAAAACATG CAAAATACCTCTAGCTCTATTGTACCT960
AGCGATCTATTTCACTATCAAGGAGATCTTGTAAAATTAGAATTTTCTACAAGAACGGAC1020
AACGATGGTCTTGCAAAAATTTTTACTGGTATTCGAAACACATTCTACAAATCGCCTAAT1080
ACTCATGAAA CATACCATGTAGATTTTAGTTATAATACCCAATCTAGTGGTAATATTTCA1140
AGAGGCTCTTCAAATCCGATTCCAATTGATCTTAATAATCCCATTATTTCAACTTGTATT1200
AGAAATTCATTTTATAAGGCAATAGCGGGATCTTCTGTTTTAGTTAATTTTAAAGA TGGC1260
ACTCAAGGGTATGCATTTGCCCAAGCACCAACAGGAGGTGCCTGGGACCATTCTTTTATT1320
GAATCTGATGGTGCCCCAGAAGGGCATAAATTAAACTATATTTATACTTCTCCAGGTGAT1380
ACATTAAGAGATTTCATCAATGTATATACTCTT ATAAGTACTCCAACTATAAATGAACTA1440
TCAACAGAAAAAATCAAAGGCTTTCCTGCGGAAAAAGGATATATCAAAAATCAAGGGATC1500
ATGAAATATTACGGTAAACCAGAATATATTAATGGAGCTCAACCAGTTAATCTGGAAAAC1560
CAGCAAACAT TAATATTCGAATTTCATGCTTCAAAAACAGCTCAATATACCATTCGTATA1620
CGTTATGCCAGTACCCAAGGAACAAAAGGTTATTTTCGTTTAGATAATCAGGAACTGCAA1680
ACGCTTAATATACCTACTTCACACAACGGTTATGTAACCGGTAATATTGGTGAAAA TTAT1740
GATTTATATACAATAGGTTCATATACAATTACAGAAGGTAACCATACTCTTCAAATCCAA1800
CATAATGATAAAAATGGAATGGTTTTAGATCGTATTGAATTTGTTCCTAAAGATTCACTT1860
CAAGATTCACCTCAAGATTCACCTCCAGAAGTT CACGAATCAACAATTATTTTTGATAAA1920
TCATCTCCAACTATATGGTCTTCTAACAAACACTCATATAGCCATATACATTTAGAAGGA1980
TCATATACAAGTCAGGGAAGTTATCCACACAATTTATTAATTAATTTATTTCATCCTACA2040
GACCCTAACA GAAATCATACTATTCATGTTAACAATGGTGATATGAATGTTGATTATGGA2100
AAAGATTCTGTAGCCGATGGGTTAAATTTTAATAAAATAACTGCTACGATACCAAGTGAT2160
GCTTGGTATAGCGGTACTATTACTTCTATGCACTTATTTAATGATAATAATTTTAA AACA2220
ATAACTCCTAAATTTGAACTTTCTAATGAATTAGAAAACATCACAACTCAAGTAAATGCT2280
TTATTCGCATCTAGTGCACAAGATACTCTCGCAAGTAATGTAAGTGATTACTGGATTGAA2340
CAGGTCGTTATGAAAGTCGATGCCTTATCAGAT GAAGTATTTGGAAAAGAGAAAAAAGCA2400
TTACGTAAATTGGTAAATCAAGCAAAACGTCTCAGTAAAATACGAAATCTTCTCATAGGT2460
GGTAATTTTGACAATTTAGTCGCTTGGTATATGGGAAAAGATGTAGTAAAAGAATCGGAT2520
CATGAATTAT TTAAAAGTGATCATGTCTTACTACCTCCCCCAACATTCCATCCTTCTTAT2580
ATTTTCCAAAAGGTGGAAGAATCAAAACTAAAACCAAATACACGTTATACTATTTCTGGT2640
TTTATCGCACATGGAGAAGATGTAGAGCTTGTTGTCTCTCGTTATGGGCAAGAAAT ACAA2700
AAAGTGATGCAAGTGCCATATGAAGAAGCACTTCCTCTTACATCTGAATCTAATTCTAGT2760
TGTTGTGTTCCAAATTTAAATATAAATGAAACACTAGCTGATCCACATTTCTTTAGTTAT2820
AGCATCGATGTTGGTTCTCTGGAAATGGAAGCG AATCCTGGTATTGAATTTGGTCTCCGT2880
ATTGTCAAACCAACAGGTATGGCACGTGTAAGTAATTTAGAAATTCGAGAAGACCGTCCA2940
TTAACAGCAAAAGAAATTCGTCAAGTACAACGTGCAGCAAGAGATTGGAAACAAAACTAT3000
GAACAAGAAC GAACAGAGATCACAGCTATAATTCAACCTGTTCTTAATCAAATTAATGCG3060
TTATACGAAAATGAAGATTGGAATGGTTCTATTCGTTCAAATGTTTCCTATCATGATCTA3120
GAGCAAATTATGCTTCCTACTTTATTAAAAACTGAGGAAATAAATTGTAATTATGA TCAT3180
CCAGCTTTTTTATTAAAAGTATATCATTGGTTTATGACAGATCGTATAGGAGAACATGGT3240
ACTATTTTAGCACGTTTCCAAGAAGCATTAGATCGTGCATATACACAATTAGAAAGTCGT3300
AATCTCCTGCATAACGGTCATTTTACAACTGAT ACAGCGAATTGGACAATAGAAGGAGAT3360
GCCCATCATACAATCTTAGAAGATGGTAGACGTGTGTTACGTTTACCAGATTGGTCTTCT3420
AATGCAACTCAAACAATTGAAATTGAAGATTTTGACTTAGATCAAGAATACCAATTGCTC3480
ATTCATGCAA AAGGAAAAGGTTCCATTACTTTACAACATGGAGAAGAAAACGAATATGTG3540
GAAACACATACTCATCATACAAATGATTTTATAACATCCCAAAATATTCCTTTCACTTTT3600
AAAGGAAATCAAATTGAAGTCCATATTACTTCAGAAGATGGAGAGTTTTTAATCGA TCAC3660
ATTACAGTAATAGAAGTTTCTAAAACAGACACAAATACAAATATTATTGAAAATTCACCA3720
ATCAATACAAGTATGAATAGTAATGTAAGAGTAGATATACCAAGAAGTCTC3771 (2)
INFORMATION FOR SEQ ID NO:2 (PS33F2): (i) SEQUENCE CHARACTERISTICS:
( A) LENGTH: 1257 amino acids (B) TYPE: amino acid (C)
STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL
SOURCE: (A) ORGANISM: Bacillus thuringiensis (C) INDIVIDUAL
ISOLATE: PS33F2 (vii) IMMEDIATE SOURCE: (B) CLONE: PS33F2a (ix)
FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..1257 (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:2:
MetAlaThrLeuAsnGluValTyrProValAsnTyrAsnValLeuSer 151015
SerAspAlaPheG lnGlnLeuAspThrThrGlyPheLysSerLysTyr 202530
AspGluMetIleLysAlaPheGluLysLysTrpLysLysGlyAlaLys 35 4045
GlyLysAspLeuLeuAspValAlaTrpThrTyrIleThrThrGlyGlu 505560
IleAspProLeuAsnValIleLysG lyValLeuSerValLeuThrLeu 65707580
IleProGluValGlyThrValAlaSerAlaAlaSerThrIleValSer 85 9095
PheIleTrpProLysIlePheGlyAspLysProAsnAlaLysAsnIle 100105110
PheGluGluLeuLysPro GlnIleGluAlaLeuIleGlnGlnAspIle 115120125
ThrAsnTyrGlnAspAlaIleAsnGlnLysLysPheAspSerLeuGln 1301 35140
LysThrIleAsnLeuTyrThrValAlaIleAspAsnAsnAspTyrVal 145150155160
ThrAlaLysThrGlnLeuGluA snLeuAsnSerIleLeuThrSerAsp 165170175
IleSerIlePheIleProGluGlyTyrGluThrGlyGlyLeuProTyr 180 185190
TyrAlaMetValAlaAsnAlaHisIleLeuLeuLeuArgAspAlaIle 195200205
ValAsnAlaGluLysLeuGlyPh eSerAspLysGluValAspThrHis 210215220
LysLysTyrIleLysMetThrIleHisAsnHisThrGluAlaValIle 225230 235240
LysAlaPheLeuAsnGlyLeuAspLysPheLysSerLeuAspValAsn 245250255
SerTyrAsnLysLysAlaAsn TyrIleLysGlyMetThrGluMetVal 260265270
LeuAspLeuValAlaLeuTrpProThrPheAspProAspHisTyrGln 275 280285
LysGluValGluIleGluPheThrArgThrIleSerSerProIleTyr 290295300
GlnProValProLysAsnMetGlnAsnThr SerSerSerIleValPro 305310315320
SerAspLeuPheHisTyrGlnGlyAspLeuValLysLeuGluPheSer 325 330335
ThrArgThrAspAsnAspGlyLeuAlaLysIlePheThrGlyIleArg 340345350
AsnThrPheTyrLysSerProA snThrHisGluThrTyrHisValAsp 355360365
PheSerTyrAsnThrGlnSerSerGlyAsnIleSerArgGlySerSer 370375 380
AsnProIleProIleAspLeuAsnAsnProIleIleSerThrCysIle 385390395400
ArgAsnSerPheTyrLysAlaIleAl aGlySerSerValLeuValAsn 405410415
PheLysAspGlyThrGlnGlyTyrAlaPheAlaGlnAlaProThrGly 420 425430
GlyAlaTrpAspHisSerPheIleGluSerAspGlyAlaProGluGly 435440445
HisLysLeuAsnTyrIleTyrThrSer ProGlyAspThrLeuArgAsp 450455460
PheIleAsnValTyrThrLeuIleSerThrProThrIleAsnGluLeu 465470 475480
SerThrGluLysIleLysGlyPheProAlaGluLysGlyTyrIleLys 485490495
AsnGlnGlyIleMetLysTyrTyr GlyLysProGluTyrIleAsnGly 500505510
AlaGlnProValAsnLeuGluAsnGlnGlnThrLeuIlePheGluPhe 5155 20525
HisAlaSerLysThrAlaGlnTyrThrIleArgIleArgTyrAlaSer 530535540
ThrGlnGlyThrLysGlyTyrPheArgLeuAspA snGlnGluLeuGln 545550555560
ThrLeuAsnIleProThrSerHisAsnGlyTyrValThrGlyAsnIle 565 570575
GlyGluAsnTyrAspLeuTyrThrIleGlySerTyrThrIleThrGlu 580585590
GlyAsnHisThrLeuGlnIleGlnHi sAsnAspLysAsnGlyMetVal 595600605
LeuAspArgIleGluPheValProLysAspSerLeuGlnAspSerPro 610615 620
GlnAspSerProProGluValHisGluSerThrIleIlePheAspLys 625630635640
SerSerProThrIleTrpSerSerAsnLys HisSerTyrSerHisIle 645650655
HisLeuGluGlySerTyrThrSerGlnGlySerTyrProHisAsnLeu 660 665670
LeuIleAsnLeuPheHisProThrAspProAsnArgAsnHisThrIle 675680685
HisValAsnAsnGlyAspMetAsnValAsp TyrGlyLysAspSerVal 690695700
AlaAspGlyLeuAsnPheAsnLysIleThrAlaThrIleProSerAsp 705710715 720
AlaTrpTyrSerGlyThrIleThrSerMetHisLeuPheAsnAspAsn 725730735
AsnPheLysThrIleThrProLysPheG luLeuSerAsnGluLeuGlu 740745750
AsnIleThrThrGlnValAsnAlaLeuPheAlaSerSerAlaGlnAsp 755760 765
ThrLeuAlaSerAsnValSerAspTyrTrpIleGluGlnValValMet 770775780
LysValAspAlaLeuSerAspGluValPheGlyLysGl uLysLysAla 785790795800
LeuArgLysLeuValAsnGlnAlaLysArgLeuSerLysIleArgAsn 805810 815
LeuLeuIleGlyGlyAsnPheAspAsnLeuValAlaTrpTyrMetGly 820825830
LysAspValValLysGluSerAspHisGlu LeuPheLysSerAspHis 835840845
ValLeuLeuProProProThrPheHisProSerTyrIlePheGlnLys 850855 860
ValGluGluSerLysLeuLysProAsnThrArgTyrThrIleSerGly 865870875880
PheIleAlaHisGlyGluAspValGluLeuVal ValSerArgTyrGly 885890895
GlnGluIleGlnLysValMetGlnValProTyrGluGluAlaLeuPro 900905 910
LeuThrSerGluSerAsnSerSerCysCysValProAsnLeuAsnIle 915920925
AsnGluThrLeuAlaAspProHisPhePheSerT yrSerIleAspVal 930935940
GlySerLeuGluMetGluAlaAsnProGlyIleGluPheGlyLeuArg 945950955 960
IleValLysProThrGlyMetAlaArgValSerAsnLeuGluIleArg 965970975
GluAspArgProLeuThrAlaLysGluIleAr gGlnValGlnArgAla 980985990
AlaArgAspTrpLysGlnAsnTyrGluGlnGluArgThrGluIleThr 9951000 1005
AlaIleIleGlnProValLeuAsnGlnIleAsnAlaLeuTyrGluAsn 101010151020
GluAspTrpAsnGlySerIleArgSerAsnValSerTyrH isAspLeu 1025103010351040
GluGlnIleMetLeuProThrLeuLeuLysThrGluGluIleAsnCys 10451050 1055
AsnTyrAspHisProAlaPheLeuLeuLysValTyrHisTrpPheMet 106010651070
ThrAspArgIleGlyGluHisGlyThrIle LeuAlaArgPheGlnGlu 107510801085
AlaLeuAspArgAlaTyrThrGlnLeuGluSerArgAsnLeuLeuHis 10901095 1100
AsnGlyHisPheThrThrAspThrAlaAsnTrpThrIleGluGlyAsp 1105111011151120
AlaHisHisThrIleLeuGluAspGlyArgA rgValLeuArgLeuPro 112511301135
AspTrpSerSerAsnAlaThrGlnThrIleGluIleGluAspPheAsp 11401 1451150
LeuAspGlnGluTyrGlnLeuLeuIleHisAlaLysGlyLysGlySer 115511601165
IleThrLeuGlnHisGlyGluGluAsnGlu TyrValGluThrHisThr 117011751180
HisHisThrAsnAspPheIleThrSerGlnAsnIleProPheThrPhe 11851190119 51200
LysGlyAsnGlnIleGluValHisIleThrSerGluAspGlyGluPhe 120512101215
LeuIleAspHisIleThrValIleG luValSerLysThrAspThrAsn 122012251230
ThrAsnIleIleGluAsnSerProIleAsnThrSerMetAsnSerAsn 12351 2401245
ValArgValAspIleProArgSerLeu 12501255 (2) INFORMATION FOR SEQ ID
NO:3 (PS52A1): (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1425 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: BACILLUS
THURINGIENSIS (C) INDIVIDUAL ISOLATE: PS52A1 (vii) IMMEDIATE
SOURCE: (A) LIBRARY: LAMBDAGEM(TM)-11 LIBRARY OF KENNETH NARVA (B)
CLONE: PS52A1-A (ix) FEATURE: (A) NAME/KEY: matpeptide (B)
LOCATION: 1..1425 (D) OTHER INFORMATION: /product="OPEN READING
FRAME OF MATURE PROTEIN" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATGATTATTGATAGTAAAACGACTTTACCTAGACATTCACTTATTCATACAATTAAATTA60
AATTCTAATAAGAAATATGGTCCTGGTGATATGACTAATGGAAATCAATTTATTATTTCA120
AAACAAGA ATGGGCTACGATTGGAGCATATATTCAGACTGGATTAGGTTTACCAGTAAAT180
GAACAACAATTAAGAACACATGTTAATTTAAGTCAGGATATATCAATACCTAGTGATTTT240
TCTCAATTATATGATGTTTATTGTTCTGATAAAACTTCAGCAGAATGGTGGAATAAA AAT300
TTATATCCTTTAATTATTAAATCTGCTAATGATATTGCTTCATATGGTTTTAAAGTTGCT360
GGTGATCCTTCTATTAAGAAAGATGGATATTTTAAAAAATTGCAAGATGAATTAGATAAT420
ATTGTTGATAATAATTCCGATGATGATGCAATA GCTAAAGCTATTAAAGATTTTAAAGCG480
CGATGTGGTATTTTAATTAAAGAAGCTAAACAATATGAAGAAGCTGCAAAAAATATTGTA540
ACATCTTTAGATCAATTTTTACATGGTGATCAGAAAAAATTAGAAGGTGTTATCAATATT600
CAAAAACGTT TAAAAGAAGTTCAAACAGCTCTTAATCAAGCCCATGGGGAAAGTAGTCCA660
GCTCATAAAGAGTTATTAGAAAAAGTAAAAAATTTAAAAACAACATTAGAAAGGACTATT720
AAAGCTGAACAAGATTTAGAGAAAAAAGTAGAATATAGTTTTCTATTAGGACCATT GTTA780
GGATTTGTTGTTTATGAAATTCTTGAAAATACTGCTGTTCAGCATATAAAAAATCAAATT840
GATGAGATAAAGAAACAATTAGATTCTGCTCAGCATGATTTGGATAGAGATGTTAAAATT900
ATAGGAATGTTAAATAGTATTAATACAGATATT GATAATTTATATAGTCAAGGACAAGAA960
GCAATTAAAGTTTTCCAAAAGTTACAAGGTATTTGGGCTACTATTGGAGCTCAAATAGAA1020
AATCTTAGAACAACGTCGTTACAAGAAGTTCAAGATTCTGATGATGCTGATGAGATACAA1080
ATTGAACTTG AGGACGCTTCTGATGCTTGGTTAGTTGTGGCTCAAGAAGCTCGTGATTTT1140
ACACTAAATGCTTATTCAACTAATAGTAGACAAAATTTACCGATTAATGTTATATCAGAT1200
TCATGTAATTGTTCAACAACAAATATGACATCAAATCAATACAGTAATCCAACAAC AAAT1260
ATGACATCAAATCAATATATGATTTCACATGAATATACAAGTTTACCAAATAATTTTATG1320
TTATCAAGAAATAGTAATTTAGAATATAAATGTCCTGAAAATAATTTTATGATATATTGG1380
TATAATAATTCGGATTGGTATAATAATTCGGAT TGGTATAATAAT1425 (2) INFORMATION
FOR SEQ ID NO:4 (PS52A1): (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:
475 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)
TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A ) ORGANISM:
BACILLUS THURINGIENSIS (C) INDIVIDUAL ISOLATE: PS52A1 (vii)
IMMEDIATE SOURCE: (A) LIBRARY: LAMBDAGEM(TM)-11 LIBRARY OF KENNETH
NARVA (B) CLONE: PS52A1-A (ix) FEATURE: (A) NAME/KEY: Protein (B)
LOCATION: 1..475 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
MetIleIleAspSerLysThrThrLeuProArgHisSerLeuI leHis 151015
ThrIleLysLeuAsnSerAsnLysLysTyrGlyProGlyAspMetThr 2025 30
AsnGlyAsnGlnPheIleIleSerLysGlnGluTrpAlaThrIleGly 354045
AlaTyrIleGlnThrGlyLeuGlyLeuProValAsnGluGlnGlnL eu 505560
ArgThrHisValAsnLeuSerGlnAspIleSerIleProSerAspPhe 65707580
SerGlnLeuTyrAspValTyrCysSerAspLysThrSerAlaGluTrp 859095
TrpAsnLysAsnLeuTyrProLeuIleIleLysSerAlaAsnAspI le 100105110
AlaSerTyrGlyPheLysValAlaGlyAspProSerIleLysLysAsp 115120125
GlyTyrPheLysLysLeuGlnAspGluLeuAspAsnIleValAspAsn 130135140
AsnSerAspAspAspAlaIleAlaLysAlaIleLysAspPheLysAla 145 150155160
ArgCysGlyIleLeuIleLysGluAlaLysGlnTyrGluGluAlaAla 165170175
LysAsnIleValThrSerLeuAspGlnPheLeuHisGlyAspGlnLys 180185190
LysLeuGluGlyValIleAsnIleGlnLysArgLeuLysGluValGln 195200205
ThrAlaLeuAsnGlnAlaHisGlyGluSerSerProAlaHisLysGlu 210215220 LeuLeuG
luLysValLysAsnLeuLysThrThrLeuGluArgThrIle 225230235240
LysAlaGluGlnAspLeuGluLysLysValGluTyrSerPheLeuLeu 245250255
GlyProLeuLeuGlyPheValValTyrGluIleLeuGluAsnThrAla 260265270
ValGlnHisIleLysAsnGlnIleAspGluIleLysLysGlnLeuAsp 275280285
SerAlaGlnHisAspLeuAspArgAspValLysIleIleGlyMetLeu 290295300
AsnSerIleAsnThrAspIleAspAsnLeuTyrSerGlnGlyGlnGlu 305310315320 Ala
IleLysValPheGlnLysLeuGlnGlyIleTrpAlaThrIleGly 325330335
AlaGlnIleGluAsnLeuArgThrThrSerLeuGlnGluValGlnAsp 340345350
SerAspAspAlaAspGluIleGlnIleGluLeuGluAspAlaSerAsp 355360365 Ala
TrpLeuValValAlaGlnGluAlaArgAspPheThrLeuAsnAla 370375380
TyrSerThrAsnSerArgGlnAsnLeuProIleAsnValIleSerAsp 385 390395400
SerCysAsnCysSerThrThrAsnMetThrSerAsnGlnTyrSerAsn 405410415 P
roThrThrAsnMetThrSerAsnGlnTyrMetIleSerHisGluTyr 420425430
ThrSerLeuProAsnAsnPheMetLeuSerArgAsnSerAsnLeuGlu 435440445
TyrLysCysProGluAsnAsnPheMetIleTyrTrpTyrAsnAsnSer 450455460
AspTrpTyrAs nAsnSerAspTrpTyrAsnAsn 465470475 (2) INFORMATION FOR
SEQ ID NO:5 (PS69D1): (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:
1185 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)
TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ii i)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)
ORGANISM: BACILLUS THURINGIENSIS (C) INDIVIDUAL ISOLATE: PS69D1
(vii) IMMEDIATE SOURCE: (A) LIBRARY: LAMBDAGEM(TM)-11 LIBRARY OF
KENNETH NARVA (B) CLONE: PS69D1A (ix) FEATURE: (A) NAME/KEY:
matpeptide (B) LOCATION: 1..1185 (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:5: ATGATTTTAGGGAATGG
AAAGACTTTACCAAAGCATATAAGATTAGCTCATATTTTTGCA60
ACACAGAATTCTTCAGCTAAGAAAGACAATCCTCTTGGACCAGAGGGGATGGTTACTAAA120
GACGGTTTTATAATCTCTAAGGAAGAATGGGCATTTGTGCAGGCCTATGTGACTACAGGC180
ACTGGTTTACCTATCAATGACGATGAGATGCGTAGACATGTTGGGTTACCATCACGCATT240
CAAATTCCTGATGATTTTAATCAATTATATAAGGTTTATAATGAAGATAAACATTTATGC300
AGTTGGTGGAATGGTTTCTTGTTTCCATTAGTTCTTAAAACAGCTAATGA TATTTCCGCT360
TACGGATTTAAATGTGCTGGAAAGGGTGCCACTAAAGGATATTATGAGGTCATGCAAGAC420
GATGTAGAAAATATTTCAGATAATGGTTATGATAAAGTTGCACAAGAAAAAGCACATAAG480
GATCTGCAGGCGCGTTGTAAAATCCTTATTAAGGA GGCTGATCAATATAAAGCTGCAGCG540
GATGATGTTTCAAAACATTTAAACACATTTCTTAAAGGCGGTCAAGATTCAGATGGCAAT600
GATGTTATTGGCGTAGAGGCTGTTCAAGTACAACTAGCACAAGTAAAAGATAATCTTGAT660
GGCCTATATGGCGACAAAAG CCCAAGACATGAAGAGTTACTAAAGAAAGTAGACGACCTG720
AAAAAAGAGTTGGAAGCTGCTATTAAAGCAGAGAATGAATTAGAAAAGAAAGTGAAAATG780
AGTTTTGCTTTAGGACCATTACTTGGATTTGTTGTATATGAAATCTTAGAGCTAACTGCG840
GTCAAA AGTATACACAAGAAAGTTGAGGCACTACAAGCCGAGCTTGACACTGCTAATGAT900
GAACTCGACAGAGATGTAAAAATCTTAGGAATGATGAATAGCATTGACACTGATATTGAC960
AACATGTTAGAGCAAGGTGAGCAAGCTCTTGTTGTATTTAGAAAAATTGCAGGCATT TGG1020
AGTGTTATAAGTCTTAATATCGGCAATCTTCGAGAAACATCTTTAAAAGAGATAGAAGAA1080
GAAAATGATGACGATGCACTGTATATTGAGCTTGGTGATGCCGCTGGTCAATGGAAAGAG1140
ATAGCCGAGGAGGCACAATCCTTTGTACTAAATGCTTATA CTCCT1185 (2) INFORMATION
FOR SEQ ID NO:6 (PS69D1): (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:
395 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)
TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM:
BACILLUS THURINGIENSIS (C) INDIVIDUAL ISOLATE: PS69D1 (vii)
IMMEDIATE SOURCE: (A) LIBRARY: LAMBDAGEM(TM)-11 LIBRARY OF KENNETH
NARVA (B) CLONE: PS69D1A (ix) FEATURE: (A) NAME/KEY: Protein (B)
LOCATION: 1..395 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
MetIleLeuGlyAsnGlyLysThrLeuProLysHisIleArgLeuAla 151015
HisIlePheAlaThrGlnAsnSerSerAlaLysLysAspAsnProLeu 202530
GlyProGluGlyMetValThrLysAspGlyPheIleIleSerLysGlu 354045
GluTrpAlaPheValGlnAlaTyrValThrThrGlyThrGlyLeuPro 505560
IleAsnAspAspGluMetArgArgHisValGlyLeuProSerArgIle 65707580 GlnIl
eProAspAspPheAsnGlnLeuTyrLysValTyrAsnGluAsp 859095
LysHisLeuCysSerTrpTrpAsnGlyPheLeuPheProLeuValLeu 100105110
LysThrAlaAsnAspIleSerAlaTyrGlyPheLysCysAlaGlyLys 115120125 GlyAla
ThrLysGlyTyrTyrGluValMetGlnAspAspValGluAsn 130135140
IleSerAspAsnGlyTyrAspLysValAlaGlnGluLysAlaHisLys 145 150155160
AspLeuGlnAlaArgCysLysIleLeuIleLysGluAlaAspGlnTyr 165170175 LysA
laAlaAlaAspAspValSerLysHisLeuAsnThrPheLeuLys 180185190
GlyGlyGlnAspSerAspGlyAsnAspValIleGlyValGluAlaVal 195200205
GlnValGlnLeuAlaGlnValLysAspAsnLeuAspGlyLeuTyrGly 210215220
AspLysSerProAr gHisGluGluLeuLeuLysLysValAspAspLeu 225230235240
LysLysGluLeuGluAlaAlaIleLysAlaGluAsnGluLeuGluLys 245250255
LysValLysMetSerPheAlaLeuGlyProLeuLeuGlyPheValVal 260265270 TyrGlu
IleLeuGluLeuThrAlaValLysSerIleHisLysLysVal 275280285
GluAlaLeuGlnAlaGluLeuAspThrAlaAsnAspGluLeuAspArg 290 295300
AspValLysIleLeuGlyMetMetAsnSerIleAspThrAspIleAsp 305310315320
AsnMetLeu GluGlnGlyGluGlnAlaLeuValValPheArgLysIle 325330335
AlaGlyIleTrpSerValIleSerLeuAsnIleGlyAsnLeuArgGlu 340345350
ThrSerLeuLysGluIleGluGluGluAsnAspAspAspAlaLeuTyr
355360365 IleGluLeuG lyAspAlaAlaGlyGlnTrpLysGluIleAlaGluGlu
370375380 AlaGlnSerPheValLeuAsnAlaTyrThrPro 385390 395 (2)
INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:7: AlaThrLeuAsnGluValTyrProValAsn 1
510 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:8:
MetIleIleAspSerLysThrThrLeuProArg HisSerLeuIleAsn 151015 Thr (2)
INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear ( ii) MOLECULE TYPE: protein (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:9:
GlnLeuGlnAlaGlnProLeuIleProTyrAsnValLeuAla 1510 (2) INFORMATION FOR
SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino
acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:10: MetIleLeuGlyAsnGlyLysThrLeuProLysHisIleArgLeuAla 151015
HisIlePheAlaThrGlnAsnSer 20 (2) INFORMATION FOR SEQ ID NO:11: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE:
amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ValGlnArgIl eLeuAspGluLysLeuSerPheGlnLeuIleLys 151015 (2)
INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 21 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:12: GCWACWTTAAATGAAGTWTAT21 (2)
INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 21 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:13: AATGAAGTWTATCCWGTWAAT21 (2)
INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 38 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:14:
GCAAGCGGCCGCTTATGGAATAAATTCAATTYKRTCWA38 (2) INFORMATION FOR SEQ ID
NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 56 bases (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:15: ATGATTATTGATTCTAAAACAACATTACCAAGACATTCWTTAATWAATACWATWAA56
(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 38 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) ( xi)
SEQUENCE DESCRIPTION: SEQ ID NO:16:
AAACATATTAGATTAGCACATATTTTTGCAACACAAAA38 (2) INFORMATION FOR SEQ ID
NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 bases (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:17: CAAYTACAAGCWCAACC17 (2) INFORMATION FOR SEQ ID NO:18: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 bases (B) TYPE: nucleic
acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: TTC
ATCTAAAATTCTTTGWAC21
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